Title: Regulation of Ca2+-dependent Desensitization in the Vanilloid Receptor TRPV1 by Calcineurin and cAMP-dependent Protein Kinase
Abstract: The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A·cyclophilin A complex (CsA·CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA·CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA·CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA·CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue. The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A·cyclophilin A complex (CsA·CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA·CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA·CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA·CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue. The capsaicin receptor TRPV1, a nonselective cation channel expressed predominantly in nociceptive sensory neurons, transduces and integrates various stimuli such as noxious heat (>42 °C), capsaicin, protons, (1.Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6940) Google Scholar, 2.Tominaga M. Caterina M.J. Malmberg A.B. Rosen T.A. Gilbert H. Skinner K. Raumann B.E. Basbaum A.I. Julius D. Neuron. 1998; 21: 531-543Abstract Full Text Full Text PDF PubMed Scopus (2559) Google Scholar), the endogenous cannabinoid anandamide (3.Zygmunt P.M. Petersson J. Andersson D.A. Chuang H. Sorgard M. Di Marzo V. Julius D. Hogestatt E.D. Nature. 1999; 400: 452-457Crossref PubMed Scopus (1844) Google Scholar), lipoxygenase products, and other lipids related to arachidonic acid (4.Hwang S.W. Cho H. Kwak J. Lee S.-Y. Kang C.-J. Jung J. Cho S. Min K.H. Suh Y.-G. Kim D. Oh U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6155-6160Crossref PubMed Scopus (960) Google Scholar) and ethanol (5.Trevisani M. Smart D. Gunthorpe M.J. Tognetto M. Barbieri M. Campi B. Amadesi S. Gray J. Jerman J.C. Brough S.J. Owen D. Smith G.D. Randall A.D. Harrison S. Bianchi A. Davis J.B. Geppetti P. Nat. Neurosci. 2002; 5: 546-551Crossref PubMed Scopus (387) Google Scholar). Studies on TRPV1 gene knock-out mice suggest that TRPV1 is essential for the development of thermal hyperalgesia following inflammation or local injection of bradykinin and nerve growth factor (6.Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Science. 2000; 288: 306-313Crossref PubMed Scopus (2868) Google Scholar, 7.Davis J.B. Gray J. Gunthorpe M.J. Hatcher J.P. Davey P.T. Overend P. Harries M.H. Latcham J. Clapham C. Atkinson K. Hughes S.A. Rance K. Grau E. Harper A.J. Pugh P.L. Rogers D.C. Bingham S. Randall A. Sheardown S.A. Nature. 2000; 405: 183-187Crossref PubMed Scopus (1474) Google Scholar, 8.Chuang H.H. Prescott E.D. Kong H. Shields S. Jordt S.E. Basbaum A.I. Chao M.V. Julius D. Nature. 2001; 411: 957-962Crossref PubMed Scopus (1065) Google Scholar).Activation of TRPV1 leads to Ca2+ influx into nociceptive sensory neurons, resulting in membrane depolarization and release of proinflammatory neuropeptides from primary afferent nerve terminals (9.Szallasi A. Blumberg P.M. Pharmacol. Rev. 1999; 51: 159-212PubMed Google Scholar). Prolonged or repeated activation of TRPV1 results in desensitization and insensitivity of the receptor to subsequent stimuli (10.Koplas P.A. Rosenberg R.L. Oxford G.S. J. Neurosci. 1997; 17: 3525-3537Crossref PubMed Google Scholar, 11.Mohapatra D.P. Wang S.Y. Wang G.K. Nau C. Mol. Cell Neurosci. 2003; 23: 314-324Crossref PubMed Scopus (75) Google Scholar). The physiological role and importance of TRPV1 desensitization is unknown but speculated to be a process of adaptation and regulation of the peripheral nervous system for the perception of pain. Comparable with other ion channels, desensitization of TRPV1 is at least in part a Ca2+-dependent process (10.Koplas P.A. Rosenberg R.L. Oxford G.S. J. Neurosci. 1997; 17: 3525-3537Crossref PubMed Google Scholar, 11.Mohapatra D.P. Wang S.Y. Wang G.K. Nau C. Mol. Cell Neurosci. 2003; 23: 314-324Crossref PubMed Scopus (75) Google Scholar). There is growing evidence for the involvement of Ca2+-dependent phosphorylation and dephosphorylation processes to regulate desensitization and excitability of TRPV1. Previous studies in rat dorsal root ganglion neurons have demonstrated that desensitization is reduced in the presence of inhibitors of the Ca2+- and calmodulin-dependent protein phosphatase 2B (calcineurin) (12.Docherty R.J. Yeats J.C. Bevan S. Boddeke H.W. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 828-837Crossref PubMed Scopus (275) Google Scholar). Conversely, phosphorylation of TRPV1 by Ca2+-calmodulin-dependent kinase II (CaMKII) 1The abbreviations used are: CaMKII, Ca2+-calmodulin-dependent kinase II; CaM, calmodulin; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; WT, wild type; CsA, cyclosporin A; CyP, cyclophilin A; CsA·CyP, cyclosporin A·cyclophilin A complex; FSK, forskolin; HEK, human embryonic kidney; PMA, phorbol 12-myristate 13-acetate; OA, okadaic acid; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride. 1The abbreviations used are: CaMKII, Ca2+-calmodulin-dependent kinase II; CaM, calmodulin; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; WT, wild type; CsA, cyclosporin A; CyP, cyclophilin A; CsA·CyP, cyclosporin A·cyclophilin A complex; FSK, forskolin; HEK, human embryonic kidney; PMA, phorbol 12-myristate 13-acetate; OA, okadaic acid; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride. seems to be a prerequisite for activation of TRPV1 by capsaicin (13.Jung J. Shin J.S. Lee S.Y. Hwang S.W. Koo J. Cho H. Oh U. J. Biol. Chem. 2004; 279: 7048-7054Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar).Another candidate involved in the mechanisms of Ca2+ negative feedback and Ca2+-dependent inactivation in many ion channels is the Ca2+ sensor calmodulin (CaM) itself. There is growing evidence that multiple regions of TRPV1 indeed may bind CaM (14.Numazaki M. Tominaga T. Takeuchi K. Murayama N. Toyooka H. Tominaga M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8002-8006Crossref PubMed Scopus (277) Google Scholar, 15.Rosenbaum T. Gordon-Shaag A. Munari M. Gordon S.E. J. Gen. Physiol. 2004; 123: 53-62Crossref PubMed Scopus (250) Google Scholar).TRPV1 is also a target for cAMP-dependent protein kinase (PKA)- and protein kinase C (PKC)-dependent phosphorylation. Phosphorylation by PKA sensitizes the channel to heat (16.Rathee P.K. Distler C. Obreja O. Neuhuber W. Wang G.K. Wang S.Y. Nau C. Kress M. J. Neurosci. 2002; 22: 4740-4745Crossref PubMed Google Scholar) and capsaicin (17.Lopshire J.C. Nicol G.D. J. Neurosci. 1998; 18: 6081-6092Crossref PubMed Google Scholar) and reduces Ca2+-dependent desensitization of capsaicin- and proton-activated currents (18.Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.T. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 19.Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Amino acids residues Ser116 and Thr370 are the major substrates for PKA-dependent phosphorylation, although other putative PKA phosphorylation sites might be involved as well. Phosphorylation by PKC sensitizes the channel to capsaicin, protons, and heat (20.Premkumar L.S. Ahern G.P. Nature. 2000; 408: 985-990Crossref PubMed Scopus (717) Google Scholar, 21.Vellani V. Mapplebeck S. Moriondo A. Davis J.B. McNaughton P.A. J. Physiol. (Lond.). 2001; 534: 813-825Crossref Scopus (443) Google Scholar, 22.Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Here, residues Ser502 and Ser800 are the major substrates for PKC-dependent phosphorylation.In the present study we investigated the influence of calcineurin on Ca2+-dependent desensitization of capsaicin- and proton-activated currents and examined the interactions of calcineurin and PKA and PKC phosphorylation pathways. We found that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis and Transient Transfection—Mutagenesis of rat TRPV1-cDNA was performed with rTRPV1-pcDNA3 by means of the transformer site-directed mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA) as described previously (19.Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Human embryonic kidney (HEK) 293t cells or cells of a human adenocarcinoma-derived cell line (HeLa cells) were transfected with wild type or mutant plasmid (0.75 or 10 μg, respectively) along with reporter plasmid (CD8-pih3m, 1 μg) by the calcium phosphate precipitation method. After incubation for 12–15 h, the cells were replated in 35-mm culture dishes. Transfected cells were used for experiments within 2–3 days. Transfection-positive cells were identified by immunobeads (anti-CD-8 Dynabeads; Dynal Biotech, Oslo, Norway). Transfection efficiency was ∼50–70% on average for TRPV1-WT and mutant channels.Chemicals and Solutions—Capsaicin (8-methyl-N-vanillyl-6-nonenamide) and cyclosporin A (CsA; both Sigma-Aldrich) were dissolved in absolute ethanol to give stock solutions of 10 mm. Forskolin (FSK; Calbiochem-Novabiochem GmbH, Bad Soden, Germany), phorbol 12-myristate 13-acetate (PMA; Calbiochem-Novabiochem GmbH), and okadaic acid (OA; Alomone Labs, Ltd., Jerusalem, Israel) were dissolved in dimethyl sulfoxide to give stock solutions of 10, 1, and 1 mm, respectively. Human brain CaM and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; both Calbiochem-Novabiochem GmbH) were dissolved in double distilled water to give stock solutions of 100 mm. Cyclophilin A (CyP; Sigma-Aldrich) was dissolved in Tris-Cl, pH 7.4, containing HEPES, 1,4-dithio-dl-threitol, phenylmethanesulfonyl fluoride, and sodium azide to give a stock solution of 20 μm. All of the stock solutions were stored at –20 °C. All of the control and test solutions were applied with a polytetrafluorethylen glass multiple-barrel perfusion system. Standard bath solutions contained 70 mm NaCl, 70 mm choline Cl, 5 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, and 10 mm glucose (adjusted to pH 7.4 with tetramethylammonium hydroxide). The NaCl/Choline Cl composition was used to reduce the amplitude of the WT currents. Choline Cl did not have any influence on WT or mutant channels. Pipette solution contained 140 mm KCl, 2 mm MgCl2, 5 mm EGTA, and 10 mm HEPES (adjusted to pH 7.4 with KOH).Electrophysiological Technique and Data Analysis—Currents were recorded at room temperature with the whole cell configuration of the patch-clamp method. Holding potential was –60 mV. Patch pipettes were pulled from boroslilicate glass tubes (TW150F-3; World Precision Instruments, Sarasota, FL) and heat-polished at the tip to give a resistance of 0.8-1.2 mΩ. The currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA), filtered at 1 kHz, and sampled at 2 kHz. pCLAMP 8.0.1 software (Axon Instruments) was used for acquisition and analysis of currents. Origin 6.1 software (OriginLab Corporation, Northampton, MA) was used to perform least squares fitting and to create figures. The data are presented as the means ± S.E. or fitted value ± S.E. of the fit. An unpaired Student's t test (SigmaStat; SSPS Science, Chicago, IL) was used to evaluate the significance of changes in mean values. p values <0.05 were considered statistically significant.RESULTSInhibition of Calcineurin Decreases Ca2+-dependent Desensitization of Capsaicin-activated TRPV1-WT Currents—TRPV1 channel exhibits desensitization in a Ca2+-dependent manner (10.Koplas P.A. Rosenberg R.L. Oxford G.S. J. Neurosci. 1997; 17: 3525-3537Crossref PubMed Google Scholar, 11.Mohapatra D.P. Wang S.Y. Wang G.K. Nau C. Mol. Cell Neurosci. 2003; 23: 314-324Crossref PubMed Scopus (75) Google Scholar). It has been suggested that a rise in cytosolic Ca2+ level caused by TRPV1 activation results in the activation of Ca2+/calmodulin-dependent protein phosphatases that mediate channel desensitization (12.Docherty R.J. Yeats J.C. Bevan S. Boddeke H.W. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 828-837Crossref PubMed Scopus (275) Google Scholar). To test this hypothesis for TRPV1-WT transiently expressed in HEK293t cells, we first studied the effect of various protein phosphatase inhibitors on Ca2+-dependent desensitization of capsaicin-activated TRPV1-WT currents, specifically on the decreasing current response to successive stimulation (tachyphylaxis). We applied a series of brief (∼5 s long) pulses of 1 μm capsaicin at 2-min intervals in Ca2+-containing solution (2 mm) and measured the current response. Under control conditions, TRPV1-WT showed strong tachyphylaxis (Fig. 1A). Most of the tachyphylaxis occurred between the first and second application, as described previously (19.Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The mean current amplitudes at the second and fourth capsaicin application were 5.1 ± 2.7 and 3.1 ± 0.4% of that of the first application, respectively (Fig. 1F). Pretreatment of cells for 10 min with 1 μm OA in the pipette solution, an inhibitor of protein phosphatase 1 and 2A, did not lead to any change in Ca2+-dependent tachyphylaxis of capsaicin-activated currents (Fig. 1B). Here, current amplitudes at the second and fourth capsaicin application were 2.5 ± 1.6 and 3.7 ± 2.4% of that of the first application, respectively (Fig. 1F). Similarly, OA was without any effect on Ca2+-dependent tachyphylaxis when used in both lower or higher concentrations (0.01–100 μm, data not shown). In contrast, tachyphylaxis was significantly decreased when cells were pretreated for 10 min with the immunosuppressive drug cyclosporin A (CsA; 14 nm) together with the "immunophilin" cyclophilin A (CyP; 17 nm) in the pipette solution (Fig. 1D). CsA and CyP are known to form a drug/immunophilin complex, which associates with and thus inhibits protein phosphatase 2B (calcineurin) (23.Jin L. Harrison S.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13522-13526Crossref PubMed Scopus (191) Google Scholar). In the presence of CsA/Cyp, current amplitudes at the second and fourth capsaicin application were 58.4 ± 6.9 and 37.2 ± 6.2% of that at the first application, respectively (Fig. 1F). This is in good agreement with previously reported results obtained in rat dorsal root ganglion neurons using similar concentration of CsA and CyP in the pipette solution (12.Docherty R.J. Yeats J.C. Bevan S. Boddeke H.W. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 828-837Crossref PubMed Scopus (275) Google Scholar). Higher concentrations of CsA (up to 1 μm) along with CyP (up to 1 μm) did not lead to any further decrease in tachyphylaxis compared with that observed with 14 nm CsA plus 17 nm CyP (data not shown). Pretreatment of cells with CsA alone up to concentrations of 100 μm in the pipette solution did not lead to any change in Ca2+-dependent tachyphylaxis of capsaicin-activated currents compared with control conditions, indicating that indeed CsA complexed to CyP is the active form that specifically targets calcineurin (Fig. 1, C and F). Pretreatment of cells for 10 min with 14 nm CsA and 17 nm CyP along with 1 μm OA in the pipette solution did not lead to any further decrease in tachyphylaxis compared with that observed with CsA·CyP (Fig. 1, E and F). Pretreatment of cells with OA, CsA, or CsA·CyP in the pipette solutions did not have any significant effect on capsaicin-activated peak currents of TRPV1-WT (Table I).Table IMean amplitudes of currents (means ± S.E.) evoked by the first capsaicin/proton application in n experiments under various experimental conditionsChannelActivatorExperimental conditionResponsenpnATRPV1-WTCapsaicin6.5 ± 1.66TRPV1-WTCapsaicin100 μm OA in pipette9.3 ± 2.66TRPV1-WTCapsaicin100 μm CsA in pipette6.8 ± 1.26TRPV1-WTCapsaicin14 nm CsA, 17 nm CyP in pipette4.8 ± 1.27TRPV1-WTCapsaicin100 μm OA plus 14 nm CsA, 17 nm CyP in pipette4.3 ± 1.26TRPV1-WTCapsaicin (30 s)7.8 ± 1.86TRPV1-WTCapsaicin (30 s)14 nm CsA, 17 nm CyP in pipette5.0 ± 1.17TRPV1-WTCapsaicin100 μm W-7 in pipette7.8 ± 1.78TRPV1-WTCapsaicin100 μm CaM in pipette8.6 ± 2.06TRPV1-WTCapsaicin10 μm FSK in extracellular buffer5.2 ± 0.99TRPV1-S116ACapsaicin8.9 ± 2.37TRPV1-S116ACapsaicin14 nm CsA, 17 nm CyP in pipette6.1 ± 1.27TRPV1-T370ACapsaicin2.3 ± 0.66<0.05TRPV1-T370ACapsaicin14 nm CsA, 17 nm CyP in pipette3.2 ± 0.86TRPV1-T144ACapsaicin2.8 ± 0.66TRPV1-T144ACapsaicin14 nm CsA, 17 nm CyP in pipette3.9 ± 1.06TRPV1-WTCapsaicin0.1 μm PMA in extracellular buffer5.3 ± 2.36TRPV1-S502A/S800ACapsaicin6.2 ± 2.36TRPV1-S502A/S800ACapsaicin14 nm CsA, 17 nm CyP in pipette4.6 ± 1.28TRPV1-WTProton3.1 ± 0.66TRPV1-WTProton14 nm CsA, 17 nm CyP in pipette4.2 ± 0.76TRPV1-WTProton (30s)6.4 ± 1.56TRPV1-WTProton (30s)14 nm CsA, 17 nm CyP in pipette4.0 ± 0.86 Open table in a new tab We also investigated responses of TRPV1-WT channels to prolonged applications (30-s) of 1 μm capsaicin in Ca2+-containing (2 mm) bath solution. TRPV1-WT currents had peak amplitudes in the range of 2.6–14.4 nA with a mean of 7.1 ± 1.8 nA. Currents reached their peak at 1.6 ± 0.5 s after beginning of activation. The currents then began to decrease rapidly during continuous capsaicin application and reached values of 0.41 ± 0.18, 0.34 ± 0.06, and 0.21 ± 0.03 nA after 10, 20, and 30 s, respectively (Fig. 2A). This type of desensitization has been termed acute desensitization, consistent with previous reports (10.Koplas P.A. Rosenberg R.L. Oxford G.S. J. Neurosci. 1997; 17: 3525-3537Crossref PubMed Google Scholar, 11.Mohapatra D.P. Wang S.Y. Wang G.K. Nau C. Mol. Cell Neurosci. 2003; 23: 314-324Crossref PubMed Scopus (75) Google Scholar, 19.Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar).Fig. 2Effect of CsA complexed to CyP on acute desensitization of capsaicin-activated TRPV1 currents. Shown are whole cell current responses to 30-s-long applications of 1 μm capsaicin in Ca2+-containing (2 mm) bath solution without (A) or with 14 nm CsA +17 nm CyP in the pipette solution (B). After a whole cell voltage clamp was established, the cells were dialyzed for 10 min before the first capsaicin application. C, the areas under the current curves were measured and normalized to an idealized, nondesensitizing current of respective size. The bars represent mean values ± S.E. * indicates a statistically significant difference compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Pretreatment of cells for 10 min with 14 nm CsA plus 17 nm CyP in the pipette solution also led to a significant decrease in acute desensitization of capsaicin-activated TRPV1-WT currents. The currents had peak amplitudes in the range of 2.4-8.9 nA with a mean of 5.0 ± 1.1 nA, which was not significantly different from control conditions (Table I). The currents reached their peak at 1.9 ± 0.7 s after beginning of activation. Then the currents began to decrease during continuous capsaicin application and reached values of 2.18 ± 0.62, 0.64 ± 0.08, and 0.59 ± 0.05 nA after 10, 20, and 30 s, respectively (Fig. 2B). To describe and compare acute desensitization quantitatively for control conditions and in the presence of CsA·CyP, we measured the areas under the current curves over a time of 30 s and normalized them to an idealized (rectangular), nondesensitizing current of a respective size. The data are given in Fig. 2C.Calmodulin Is Not Required for the Decrease in Ca2+-dependent Desensitization by Inhibition of Calcineurin—Calcineurin exerts its phosphatase activity in a Ca2+- and calmodulin-dependent manner. CaM itself is a dominant Ca2+ sensor for Ca2+-dependent inactivation in many ion channels (24.Lee A. Wong S.T. Gallagher D. Li B. Storm D.R. Scheuer T. Catterall W.A. Nature. 1999; 399: 155-159Crossref PubMed Scopus (995) Google Scholar, 25.Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (731) Google Scholar, 26.Trudeau M.C. Zagotta W.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8424-8429Crossref PubMed Scopus (50) Google Scholar). There is accumulating evidence that multiple regions of TRPV1 may bind CaM. One putative region was identified in the C-terminal (14.Numazaki M. Tominaga T. Takeuchi K. Murayama N. Toyooka H. Tominaga M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8002-8006Crossref PubMed Scopus (277) Google Scholar), and another was identified in the N-terminal segment (15.Rosenbaum T. Gordon-Shaag A. Munari M. Gordon S.E. J. Gen. Physiol. 2004; 123: 53-62Crossref PubMed Scopus (250) Google Scholar). We investigated the functional role of CaM in Ca2+-dependent desensitization of capsaicin-activated TRPV1-WT channels. The experiments were performed as described for those shown in Fig. 1. Pretreatment of cells with 100 μm W-7 in the pipette solution, which is a potent noncompetitive antagonist of calmodulin, did not lead to any change in Ca2+-dependent tachyphylaxis of capsaicin-activated TRPV1-WT currents (Fig. 3, A and C). W-7 also did not have any significant effect on capsaicin-activated peak currents of TRPV1-WT (Table I). This is in good agreement with an earlier report about the ineffectiveness of W-7 on TRPV1 channel desensitization (14.Numazaki M. Tominaga T. Takeuchi K. Murayama N. Toyooka H. Tominaga M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8002-8006Crossref PubMed Scopus (277) Google Scholar). Tachyphylaxis was significantly decreased when cells were pretreated with 100 μm W-7 along with 14 nm CsA plus 17 nm CyP in the pipette solution (Fig. 3B). Here, current amplitudes at the second and fourth capsaicin application were 71.7 ± 12.0 and 42.2 ± 8.1% of that of the first application, respectively (Fig. 3C). The decrease in channel tachyphylaxis was comparable with that observed in the presence of CsA plus CyP in the pipette solution (Figs. 1F and 3C). This indicates that inhibition of calcineurin alone is sufficient to inhibit TRPV1-WT channel desensitization. CaM alone up to concentrations of 100 μm in the pipette solution did not lead to any change in tachyphylaxis of capsaicin-activated TRPV1-WT currents compared with control conditions (Fig. 3, D and F). Pretreatment of cells with CaM up to concentrations of 100 μm along with 14 nm CsA and 17 nm CyP in the pipette solution led to a significant decrease in tachyphylaxis of capsaicin-activated TRPV1-WT currents (Fig. 3E). Here, the results were quantitatively similar to those obtained with CsA·CyP alone or CsA·CyP along with W-7 in the pipette solution (Figs. 1F and 3, C and F).Fig. 3Effect W-7, a calmodulin antagonist, and CaM on Ca2+-dependent desensitization of capsaicin-activated TRPV1 currents. Shown are whole cell current responses of TRPV1 channels to repeated brief (∼5-s-long) applications of 1 μm capsaicin in Ca2+-containing (2 mm) bath solution with 100 μm W-7 (A) 100 μm W-7 plus 14 nm CsA/17 nm CyP (B) 100 μm CaM (D) or 100 μm CaM plus 14 nm CsA/17 nm CyP in the pipette solution (E). After a whole cell voltage clamp was established, the cells were dialyzed for 10 min before the first capsaicin application. The intervals between capsaicin applications were 2 min. C and F, The mean amplitudes of currents ± S.E. measured in experiments as described for A and B or for D and E, respectively. The amplitudes were normalized to the current amplitude obtained with the first capsaicin application. * indicates a statistically significant difference in the mean amplitudes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phosphorylation by PKA and Dephosphorylation by Calcineurin Regulates Ca2+-dependent Desensitization of TRPV1—In the resting state, TRPV1 is highly phosphorylated, at least when heterologously expressed in CHO-K1 cells (18.Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.T. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). PKA is able to phosphorylate TRPV1. However, PKA phosphorylation only becomes obvious in the desensitized state (18.Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.T. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Phosphorylation by PKA partly rescues TRPV1 from desensitization (18.Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.T. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 19.Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Because inhibition of calcineurin decreases desensitization of capsaicin-activated TRPV1 currents to a similar extent like activation of PKA, we investigated the interplay between PKA activation and calcineurin inhibition and their effect on Ca2+-dependent desensitization of capsaicin-activated TRPV1 currents. As demonstrated previously, pretreatment of cells for 10 min with 10 μm FSK, an activator of adenylate cyclase and thus an indirect PKA activator, led to a significant decrease in channel tachyphylaxis (Fig. 4A) (19.Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Current amplitudes at the second and fourth capsaicin application were 62.7 ± 7.8 and 42.8 ± 9.8% of that at the first application, respectively (Fig. 4C). FSK pretreatment did not have any significant effect on capsaicin-activated peak currents in TRPV1-WT (Table I). Pretreatment of cells for 10 min with 14 nm CsA plus 17 nm CyP in the pipette solution along with 10 μm FSK in the external solution did not lead to any significant further decrease in tachyphylaxis compared with that in the presence of 10 μm FSK alone (Fig. 4, B and C). To exclude the possibility that the decrease in desensitization in the presence of CsA·CyP is caused by PKA phosphorylation caused by disinhibition of PKA by CsA·CyP, we investigated the effect of CsA·CyP on channel tachyphylaxis in the presence of the PKA inhibitor KT5720. In these experiments the decrease